DESCRIPTION: The long term goal of the research is to understand how the DNA replication apparatus responds to DNA template damage and how mutations occur. The applicant has developed an in vitro assay system in which replication of UV damaged DNA templates by human cell extracts results in the same types of altered patterns of DNA replication and the same spectrum of mutations as those recovered from in vivo systems. In this application she will (1) test specific models for the mechanisms of mutagenic bypass of DNA template damage and (2) use purified DNA replication factors to determine the biochemical requirements for mutation fixation. Studies to evaluate specific models for replicative bypass will: a) evaluate efficiency of bypass of lesions in the leading versus lagging strand, b) evaluate coordination of synthesis of the two replication forks, c) determine if gaps exist in newly replicated molecules and if they are filled, d) determine the source of the template for the lesion bypass, and e) establish the relationship of sequence specificity of bypass and observed mutational spectra. Studies to determine which components of a reconstituted DNA replication system are required to bypass lesions and fix mutations will be done in collaboration with Dr. John Turchi who is experienced in the characterization of the components of the in vitro replication system. The studies will: a) determine if the reconstituted system can replicate a damaged template, b) determine if mutations are fixed during replication and is the spectrum comparable to the unfractionated system, c) determine if the mechanism of replicative bypass is similar/different than that of the unfractionated cell lysate system, and d) if the reconstituted system cannot bypass the lesions, identify the component(s) lacking in the reconstituted system essential for the replicative bypass/mutation induction.